Method for the rapid detection of microorganisms in samples

ABSTRACT

The invention relates to a method for the rapid detection and enumeration of microorganisms. The spectroscopic detection is carried out, after brief incubation of the sample to be tested, by the detection of the growth of microcolonies, which is not yet detectable with the naked eye, on a solid culture plate following direct transfer of said microcolonies to a sample carrier by special impression techniques; the detection itself is carried out by recording the spectra of all, or of a representative number, of the impressions, using an IR microscope. It is not only possible to make statements with regard to the number of microorganisms in the sample but the high information content of the spectra can also be used for demanding microbiological differentiation work down to species identification.

The invention relates to a method for the rapid detection ofmicroorganisms in a multiplicity of samples, such as, for example,urine, blood, water, foodstuffs, pharmaceutical raw materials andproducts, such as, for example, ointments, liquid preparations, etc.,which as a rule should be free from such microorganisms or for whichcertain, specified limiting values for the total germ count or forindividual, defined representatives of microorganisms should not beexceeded, as well as a device for carrying out the method.

Detection work of this type is carried out on a large scale in theclinical sector (sepsis, urinary tract infections, liquor testing) andnon-clinical sector (assessment of the freedom from germs or thecontamination of starting materials and end products, such as, forexample, water, milk, foodstuffs, etc.), since the results of such testsare of great clinical significance but also of great significance forquality control, stability assessment and other product and productionmethod tests, for example in industrial sectors. Because of the frequentpossibility of exponential growth of the microorganisms to be detectedand of the very early detection of, for example, infections, which isoften necessary in the clinical sector to instigate therapeuticmeasures, for example in the case of life-threatening sepsis, or in thenon-clinical sector, for example in the case of the production ofpharmaceuticals, in order to reduce the unusability of entire batches,for example in the case of the use of contaminated starting materials,with possibly considerable economic consequences, a detection even oflow germ counts (for example <10⁵ per ml or g of sample) within as shortas possible a time is required.

In the current state of the art, the methods used for carrying outdetection tests are as a rule either methods which permit very rapid andhighly specific detection of very small amounts of specific individualgroups of microorganisms or methods which are able to detect a largevariety of different microorganisms non-specifically, less rapidly andwith comparatively distinctly larger amounts of microorganisms(approximately >10⁵ bacteria per ml of test sample) (for a reviewcompare, for example: Bergan, T. in: Methods in Microbiology, Vols.14-16, London, Orlando, San Diego, San Francisco, New York, Toronto,Montreal, Sydney, Tokyo, Sao Paulo: Academic Press (1984); Kipps, T. J.,Herzenberg, L. A., in: Habermehl, K.-O.: Rapid Methods and Automation inMicrobiology and Immunology, Berlin, New York, Tokyo: Springer Verlag(1985); Toorova, T. P., Antonov, A. S., in: Colwell, R. R. andGrigorova, R.: Methods in Microbiology, Vol. 19, London, Orlando, SanDiego, New York, Austin, Boston, Sydney, Tokyo, Toronto: Academic Press(1987); Thronsberry, C., in: Habermehl, K.-O.: Rapid Methods andAutomation in Microbiology and Immunology, Berlin, New York, Tokyo:Springer Verlag (1985); Carlberg, D. M., in: Lorian, V.: Antibiotics inLaboratory Medicine, Baltimore, London, Los Angeles, Sydney: Williams &Wilkins (1986)). The first group includes in particularmolecular-biological detection methods, with which the detection takesplace indirectly by detection of a reaction of the sample, for examplewith monoclonal antibodies or also by DNA/DNA hybridization and similartechniques. A characteristic feature of these techniques is thenecessity for the (expensive) production and preparation of highlyspecific reagents for each individual type of microorganism to bedetected. It is, however, an advantage of these methods that a detectionof the microorganisms at the same time already comprises anidentification--accurate to a greater or lesser extent depending on thespecificity of the reagents used--of the type of microorganism detected,insofar as cross-reactions with other types of organism, which are afrequent problem, can be disregarded. The use of these methods, which inany case is hardly routine to date, is therefore restricted for theforeseeable future to individual, particularly important (for examplelife-threatening germs in the case of an infection) groups of organisms.Representatives of the second group of methods typically include, forexample, microcalorimetric, conductivity/impedance and opticalmeasurements.

In the case of these methods the sample to be tested for the presence ofmicroorganisms, for example bacteria, is as a rule placed in a suitableculture medium promoting the growth of the organisms and said medium isincubated under growth conditions which are as optimum as possible.Detection then takes place, for example, by detection of the heatproduction of the culture, using a microcalorimeter, or of the changesin the impedance of the culture liquid caused by the growth of theculture, using suitable conductivity measurement systems. In the broadersense, this group of methods also includes simple optical measurementsof the turbidity of the culture medium or the light-optical detection ofmicroorganisms. The advantage of this group of methods lies in therelatively universal application to very many groups of organisms and,with the exception of the last mentioned method variant, in the factthat said methods can be operated and automated relatively simply. Adisadvantage, on the other hand, is the lack of a possibility forparallel identification of the microorganisms, which, if necessary, mustbe carried out separately using other methods, and also the relativelylong period, caused inter alia by the culture, between sampleintroduction and detection and the comparatively high detection limits(for example about 10⁵ cells/ml of urine in the case of the urologicalsamples which are particularly frequent in medical practice).

DE-A1-32 47 891 describes a method for the identification ofmicroorganisms on the basis of image-analytical evaluation of infra-redspectra in selected, small wavenumber regions (compare also Giesbrecht,P., Naumann, D., Labischinski, H., in: Habermehl: Methods and Automationin Microbiology and Immunology, pp. 198-206, Berlin, Springer Verlag(1985); Naumann, D., Fijala, V., Labischinski, H., Giesbrecht, P., J.Mol. Struct. 174, 165-170 (1988)). Despite powerful infra-redspectroscopes and infra-red microscopes, the sensitivity of thesemethods is not sufficient to enable samples to be investigated for thepresence of microorganisms.

Now, it is the object of the present invention to develop a method forthe rapid detection of a very small number of microorganisms of theabove-mentioned type and to provide the possibility for rapid andreliable detection of very diverse microorganisms, even if the number ofmicroorganisms in the sample to be tested is very small.

This object is achieved according to the invention by applying a sampleof microorganisms to an essentially solid culture carrier, in a dilutionthat enables the growth of the microorganisms into locally separatedcolonies, initiating a short-term growth of the microorganisms of atleast six generation times, transferring regions of the surface of theculture carrier on which microcolonies are present so as to maintaintheir relative distances from each other to an optical carrier that iseither at least partially transparent or reflective in the desiredspectral region, carrying out directly the transfer of themicroorganisms from the culture carrier onto a stamp, the stamp surfaceof which consists of a material that is transparent or reflective in thedesired wave range, locating individual colonies of the transferredmicroorganisms under an infrared (IR) microscope, and sequentiallyrecording and evaluating individually the IR spectra of the transferredmicroorganism colonies by means of the IR microscope in the transmissionor reflection mode, preferably using apertures in the diameter range ofabout 10-200 μm. The invention enables the advantages of the variousgroups of methods employed hitherto, that is to say detection of lowgerm counts of microorganisms, possibility for combination withidentification methods, short time requirement and broad applicabilityto as far as possible all types of organisms in accordance with auniform method which is easy to automate, to be achieved in combinationin a single method. At the same time, the method according to theinvention can also be employed effectively in routine operation inmicrobiological laboratory practice. The spatially separated growth ofmicrocolonies, starting from single individuals of the microorganismsdirectly from the sample to be tested, on the culture carrier and theexamination of a surface sample, obtained from the culture carrier,after locality-true transfer, makes the method according to theinvention suitable and valuable specifically for the examination ofsamples which per se should be free from microorganisms or which haveonly a very low germ count.

The growth period of at least 6 generation times of the organisms to bedetected, which is provided according to the invention, is based on theconsideration that a locality-bound concentration of the particularmicroorganism on a solid carrier for the sample, in particular on anagar plate, represents an enrichment of the microorganism to a degreesuch that the colonies formed are sufficiently large for detection usinga microscope, in particular an IR microscope and an IR spectroscope. Asa result of the low concentration of the microorganisms in the sample tobe tested, if necessary after a possible dilution, it is ensured thatthe individual microorganisms are applied to the surface of the solidcarrier as individuals with a spatial separation which is so large thatthe colonies of microorganisms formed therefrom do not mutually impairone another. In this way, a pure culture of the microorganisms in themicroscopic range is carried out directly from the sample, without aprior or subsequent isolation of the individual microorganism beingrequired. The size of the colonies of the microorganisms should be suchthat the particular colony has a superficial extent which corresponds toa number of at least about 50 individuals, for example bacteria.Starting from a single cell, 2⁶, or 64, cells are formed in 6 generationtimes, the superficial extent of which cells already suffices forinfra-red spectroscopy. In the case of particularly large cells, smallernumbers of cells, such as 2⁵, can also be sufficient. For safetyreasons, however, the procedure is as a rule carried out using a largersuperficial extent of the colony, which is achieved, in particular, byat least 7-12 generation times. 10 generation times correspond to about1000 cells.

The time period within which the colony growth is achieved depends onthe growth rate of the individual microorganisms. Usually, a generationtime of 20-40 minutes suffices for samples which are taken from theclinical sector and a generation time of about one hour for watersamples. In order to be sure, the period is kept at about 4-10 hours inpractice. The period which is required and suitable in a particular casecan also be determined by preliminary experiments. The result of this isthat in the case of routine use of the method it is possible to workwith the shortest possible period in order to obtain the desired resultas rapidly as possible. As a result of the locality-true transfer ofsurface layers of the resulting microcolonies to the optical carrier,mixing of constituents of different colonies is avoided. This makes themethod according to the invention also suitable for the enumeration andidentification of the microorganisms. A high optical resolution andhighly meaningful results are achieved by the use of apertures ofsuitable diameter.

A particularly suitable device for carrying out the method according tothe invention is disclosed herein. Advantageous further developments andfeatures of the method according to the invention and of the device willbecome apparent from the description which follows. The individualfeatures may be implemented individually or in any desired combinationof several or all of them.

The preferred use of infra-red microscopes, which, as a result of thefurther development of the measurement techniques for Fourier transforminfra-red spectroscopy, enable sample amounts of down to below thenanogram range to be measured reproducibly, is particularlyadvantageous. As a result of the newly developed techniques for sampletransfer of micro-colonies of the microorganisms without prior pureculture, in particular directly onto infra-red-transparent or reflectivecarrier materials, which can be measured directly in the optical andinfra-red-spectroscopic mode of the IR microscope, a particularly rapidand reliable detection is facilitated. Surprisingly, the detection issuccessful even in the case of the preferred use of one or more evenvery small sections (for example only a few 100 wavenumbers) from thetotal spectrum, so that it is also possible to use carrier materialswhich are IR-transparent or reflect well only in very small wavenumberranges. A procedure which proved particularly advantageous for rapiddetection was the use of a stamp impression method for sample transferfrom a culture carrier customarily used in bacteriology, in particularan agar culture plate, according to which method, in particular, astamp, for example a circular stamp, of IR-transparent material ofsuitable thickness is pressed briefly (for example for less than 1second) onto the culture plate--in an appropriate manner using a guide,or optionally also directly by hand--and lifted off again, whereby partsof the microcolonies of the --maybe even different--microorganisms which(after brief incubation of, for example, a Urine sample) are present onthe culture plate and which as a rule are not yet detectable with thenaked eye, can be transferred locality-true to the stamp plate, so thatthe impression samples on the stamp plate can be measured directly,without further preparation effort, on the sample stage of the FT-IRmicroscope. This procedure offers the additional advantage that, at thewish of the operator, the incubation of the original culture plates canbe continued for subsequent monitoring purposes. The transfer to thestamp used as optical carrier can also be effected by pressing themovable culture carrier against the stamp surface.

An additional advantage has been found to reside in the fact that themicroscope, in particular the light-optical mode of a preferentiallyused IR microscope (if appropriate supported by methods for imageevaluation using, for example, an attached television camera with orwithout further image processing facilities), can, if desired, also beused not only to drive the regions of the stamp which are of interestbut also for pre-evaluation of the sample (for example according to themorphology of the microcolonies, color and shape of the microorganisms(cocci, rods, etc.)). The actual, reliable detection is then carried outby recording the corresponding infra-red spectra, which, because of thefundamental chemical composition common to all microorganisms (cellwall, membrane, protein, ribosomes, nucleic acids, etc.), in principlehave similar spectral characteristics for all possible microorganisms,so that the method, as desired, can be used for practically allmicroorganisms without limitation. A particular advantage of the methodproves to be the fact that the IR-spectra of micro-organisms,irrespective of the fact that they are similar in principle as aconsequence of the specific chemical composition of the cells ofindividual types of microorganisms, if desired also permit, withoutfurther expenditure on equipment, a differentiation and identificationof microorganisms measured to be carried out by comparison of thespectra with those of a suitable reference database, and this down tothe sub-species level, using a procedure described earlier (Giesbrecht,P., Naumann, D., Labischinski, H., in: Habermehl: Methods and Automationin Microbiology and Immunology, pp. 198-206, Berlin: Springer Verlag(1985); Naumann, D., Fijala, V., Labischinski, H., Giesbrecht, P., J.Mol. Struct. 174, 165-170 (1988)), to which reference is made here.

According to the invention it is possible, as already mentioned, tocarry out an identification of microorganisms in addition to thedetection. The device according to the invention can, however,advantageously be designed such that all three main tasks, for exampledetection, identification and where appropriate even sensitivity tests,can be carried out, so that an old aim--which, however, it is notpossible to achieve with the state of the art to date--of a uniformtechnique with a procedure which is uniform in principle can now beachieved for any desired microorganisms. In this context, it proves tobe highly advantageous fully to automate the entire procedure, fromsample preparation via sample measurement to output of the result, ifdesired even including a subsequent further processing of the result,say in the sense of an identification or sensitivity test, since themethod can in principle be carried out in a uniform manner for allmicroorganisms under consideration and the technique of recordingspectra using the FT-IR microscope technique provides from the outset ameasurement result which is digital and thus easy to process byelectronic means.

When carrying out a preferred embodiment of the method according to theinvention, the sample to be tested, such as, for example, urine, blood,liquor, water, aqueous extracts from foodstuffs, ointments, medicamentsand the like, is coated in the manner customary in microbiology onto asolid culture plate in the manner known from microbiology in a dilutionsuch that the microorganisms which may be present in the sample Growindividually into colonies. After a short culture time of about 4-10hours (depending on the type of sample and the Generation time of themicroorganisms to be detected), in which microcolonies which consist,for example, of about 10³ organisms and are not yet detectable with thenaked eye have formed on the plate, the transfer from the culture plateto the carrier, which is transparent or reflective in the desiredwavenumber range, is effected, for example by placing a sample carrierdisc, which is, for example, about 2 mm thick and 3 cm in diameter andis made, for example, of ZnSe, polished steel or the like, on the plateand then lifting it off again, so that a locality-consistent impressionof adhering microorganisms from the culture plate is obtained on thesample carrier. The sample carrier is then transferred manually or underautomated control to the sample stage of the microscope, in particularan IR microscope. At this point in the method, several variants arepossible depending on the wishes of the person carrying out theexperiment and the given detailed features of the microscope. In thesimplest case, the sample plate can be manually/visually inspected usingthe optical mode of the microscope. An initial estimate of the germcount present in the sample can already be made from the number ofoptically discernible microcolonies (taking into account any dilutionfactors depending on the sample dilution in the manner customary inmicrobiology) and, if appropriate, a rough estimate can also already bemade from the colony morphology (size, color, surface nature etc.) withregard to possibly different groups of organisms. If no microcoloniesare detectable at this point, the method can already be concluded atthis point as having a "negative" result. The actual detection is thencarried out using the infra-red-spectroscopic mode of the microscope, inwhich the infra-red spectra of the corresponding microcolonies arerecorded successively, with the aid of the aperture system--which is anintegral feature of every IR microscope--for all, or for arepresentative number of, the microcolonies present on the samplecarrier. Of the microcolony impressions previously detected optically,only those which have spectra which are compatible with the chemicalcomposition typical for microorganisms (in particular proteins, lipids,nucleic acids, polysacharides) are accepted as conclusively detected. Inthis way it is prevented that areas of the impression which are detectedin the optical mode and which originate, for example, from inorganiccontamination (dust etc.) or, for example, from transferred componentsfrom the medium or agar, erroneously contribute to the result. In thesense of one process variant, the resulting spectra can, if desired,also be used immediately or at a later time with a view to identifyingthe microorganisms, by comparison with a reference spectra database inthe desired spectral region(s), which may also be very small, inaccordance with the published principles of the procedure (see above). Anumber of further method modifications are possible in order to achieve,if desired, greater independence of the method from human interventionin the procedure, as described above in principle. Thus, for example,the transfer steps provided (that is to say a) application of the sampleto the culture plate, b) transfer of the inocculated culture plate to anincubator, c) stamp impression for transfer of the microcolonies fromthe culture carrier to the optical sample carrier, d) if appropriatetransfer of the sample carrier to the sample stage of the IR microscopeand e) removal of the sample after measurement has been carried out) caneasily be mechanized and/or automated with the aid of customarymechanical and/or electronic components. A fully automated proceduregoing beyond this can be achieved by adding an image processing systemconsisting of a camera flange-mounted on the IR microscope, anelectronically controlled stage with X and Y movements as the microscopestage, an electronically controlled unit for switching between theoptical and the IR microscope mode of the microscope, a control unit orcomputing unit (for example personal computer with appropriateinterfaces or other computer systems) and one or more appropriatecomputation programs. Appropriately, the computer and control unitincluding computation programs can be integrated in the computer/controlunit which is present from the outset and serves to control the FT-IRequipment including the IR microscope.

A fully automated procedure is then as follows: after transfer of thesample carrier, containing the microcolony impressions, to the X-Y stageof the IR microscope, an image of the carrier can be recorded by thecamera in the optical mode of the microscope and supplied to thecomputer/control unit in digital form (in at least 256×256 pixels, forexample 64 grey scales). The X-Y positions of possible microcolonyimpressions can be determined in the computer/control unit using asub-program, by evaluation of the digital grey scale distribution of theimage, for example using a peak search algorithm. The control unit canthen set a suitable aperture (diameter for example 30-80 μm), carry outpositioning to the location coordinates of the first suspectedmicrocolony impression and record the infra-red spectrum at thislocation. This operation can be repeated until it has been completed forall previously determined X-Y positions. The control unit can then callup a further sub-program which checks all spectra recorded in this wayfor compatibility with microorganism spectra. This can be achieved, forexample, via a simple cross-correlation, if appropriate with priorfiltering, of the spectra with reference spectra of microorganisms,since for each individual or combined spectral region, for a givenfilter method, a threshold value of the correlation coefficient betweenmicroorganism spectra exists below which the suspected microcolony isidentified as inorganic or organic contamination of the sample carrier.With this procedure a reference database of 5-20 typical microorganismspectra (for example Staphylococci, Streptococci, Clostridia,Pseudomonads, Entero-bacteria, Salmonella, Legionella and Bacilli)suffices for reliable discrimination between microorganisms andcontamination. Of course, peak tables or, for example, evaluation usingknown methods of multi-variance statistics can also be used for specialapplications. The number of microorganisms present in the sample canthen be calculated, for example as germ count/ml of sample, by thecontrol/computer unit from the number of positions confirmed asmicrocolony impressions, if appropriate taking account of correspondingdilution factors, and can be given as output. With the aid of thecontrol and computer unit it is, of course, also possible, if desired,with appropriate programming, to carry out a subsequent identificationof the microorganisms detected, by comparison with a reference databasewhich consists of reference spectra measured under the same conditions.The parts of the program required are based on algorithms well known tothose skilled in the art and can be written from scratch or taken from amultiplicity of generally obtainable program systems (a program whichhas been successfully used in this context is, for example, the IMAGICprogram, cf. M. van Heel, W. Keegstra, Ultramicroscopy 7, 113, 1981).

Overall, the new method for the detection of microorganisms permits, inthe manner described, a rapid qualitative and quantitative detection ofmicroorganisms, in principle of all types, with the further advantage ofthe additional identification using the same basic equipment within aperiod of about 4-10 hours or less after submission of the sample,without prior pure culture, this period being determined virtually intotal by the incubation time necessary to obtain microcolonies, sincethe further process steps take only minutes to less than 1 hour if themethod is carried out in a suitable manner.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a and 1b collectively depict a flow chart showing, in theright-hand column, the general overall procedure for the rapid detectionof microorganisms, and in the left-hand column, the subcomponents of theapparatus in the situation of an automated procedure.

FIG. 2 depicts spectral plots recorded in the optical mode of an IRmicroscope of 10⁴, 10³ and 10² Klebsiella pneumoniae cells grown on anagar plate and subsequently transferred to a sample carrier.

FIG. 3 is a photograph of a section of a sample carrier which containsthree transferred Staphylococcus aureus microcolonies obtained byapplying the sample carrier to an agar plate containing themicrocolonies.

FIG. 4 depicts spectral plots, recorded in the optical mode of an IRmicroscope, for the three transferred Staphylococcus microcolonies shownin FIG. 3.

FIG. 5 is a photograph of a section of a sample carrier containing atransferred Staphylococcus aureus microcolony, a transferredStaphylococcus xylosus microcolony and a transferred Klebsiellapneumoniae microcolony obtained by applying the sample carrier to anagar plate containing the microcolonies.

FIG. 6 depicts spectral plots recorded in the infra-red mode of an IRmicroscope for the three transferred Staphylococcus aureau,Staphylococcus xylosus and Klebsiella pneumoniae microcolonies shown inFIG. 5.

EXAMPLE 1

This Example 1, in conjunction with FIG. 2, is intended to verify theexceptional sensitivity of the new method, without describing the entireprocedure.

FIG. 2 shows three spectra obtained in the following way using a A 560infra-red microscope from Bruker, Karlsruhe, coupled with an IFS 48FT-IR spectrometer from the same company:

100 μl of a 10³ germ/ml culture of Klebsiella pneuminiae, ATCC 13882 inpeptone water was coated onto a peptone agar plate and incubated for 8hours at 37° C. (about 24 generation times). The impression of thebacteria was obtained by carefully laying on and removing again apolished BaF₂ sample carrier 40 mm in diameter and 3 mm thick. Thebacterial film which is transferred in this way and as a rule(especially in the case of relatively short incubation times) consistsof 1-3 layers of bacteria, dries within a few seconds under ambientconditions without using vacuum or dessicants. Inspection of the samplecarrier in the optical mode of the IR microscope (15× lens,Casse-granian, 20× eyepiece) gave an extended, thin 1-3 layer bacterialfilm over virtually the entire sample carrier surface. By selection ofsuitable circular apertures it was achieved that in each case about 10⁴(spectral plot 1 in FIG. 2, 8 μm diameter aperture), about 10³ (spectralplot 2, 40 μm diameter aperture) and about 10² (spectral plot 3, 20 μmdiameter aperture) microorganisms contribute to the spectrum. Allspectra were recorded in transmission. 512 scans were accumulated at aspectral resolution of 8 cm⁻¹. Inspection of FIG. 2 clearly shows thatspectral signals clearly detectable as microorganism spectra can alreadybe obtained, in this case in the wavenumber range of about 4000-900,with about 10² microorganisms (corresponding to less than 0.1 ng ofsubstance). Because of the relatively high noise content (cf. spectralplot 3 in FIG. 2), however, working with at least about 10³microorganisms is to be preferred if an identification, for examplespecies identification, which is as exact as possible is to follow thedetection using the same set of data. It can be seen from this typicalmeasurement example that the incubation time of the sample on theculture plate should be at least about 6 to 12 generation times in orderto allow a microcolony of about 200-4000 organisms to form from eachmicroorganism isolated by application in appropriate dilution to theculture plate. Thus, for example, a lower limit for the incubationperiod of less than 4 hours results for Enterobacteriaceae (typicalgeneration time on customary media about 20 minutes) and of about 5hours for Staphylococci (generation time on customary media about 30minutes), etc..

EXAMPLE 2

This example shows a typical procedure in the case of an experimentcarried out manually using as an example an aqueous sample to which 10³cells/ml of Staphylococcus aureus, Pelzer strain, were artificiallyadded. 100 μl of this sample were applied to a peptone agar plate (100mm diameter) and distributed with the aid of a Drigalski spatula. Afteran incubation time of 8 hours at 37° C. in an incubator, themicrocolonies (not yet discernible with the naked eye) were transferredlocality-true to a 60 mm BaF₂ plate in the manner described in Example 1(stamp impression).

FIG. 3 shows a photograph (magnification 250) of a section of the samplecarrier. The spectra obtained in the region between 1800 and 800 cm⁻¹for the three microcolony impressions shown in FIG. 3 are shown in FIG.4. Visual comparison of the three spectra already shows the excellentreproducibility of the method in detecting different microcolonies ofthe same microorganism. For quantitative confirmation, the so-calleddifferentiation index D (D=(1-α)·1000, α=Pearson correlationcoefficient) was determined by cross-correlation. In the case of themeasurements shown here and also of measurements carried out analogouslyfor further species a D value of ≈8 was obtained, incorporatingmeasurements on independently prepared samples which were prepared atdifferent times on agar plates of different batches of the same nutrientmedium. Thus, the D value of about 8 (corresponding to a correlationcoefficient of 0.992!) defines the surprisingly good level ofreproduction of the method and provides the threshold value fordifferentiation of different microorganisms in the case where subsequentdifferentiation or identification of the detected microorganisms isdesired. In the example described, 17 microcolony impressions weredetected on the carrier plate by means of their infra-red spectrum.Taking account of the amount of sample employed (100 μl) and of theratio of the surface area of the agar plate to the surface area of thesample carrier (6.26:1), a detected germ count of 1.06×10³, which is inexcellent agreement with the value employed, is calculated from thisvalue.

EXAMPLE 3

This example illustrates the procedure for identification of themicroorganisms found, following their detection: 100 μl of a watersample containing Staphylococcus aureus, Staphylococcus xylosus andKlebsiella pneumonias, in each case in an amount of 10³ germs per ml,were applied as in Example 2 to a peptone/agar plate and after anincubation time of 8 hours applied to the same sample carrier asdescribed in Example 2, using the identical stamp technique.

FIG. 3 shows a section of the sample carrier, with a magnification of250. Because of the locality-true and thus area-true transfer of themicrocolonies, observation in the optical mode of the microscope alreadyindicates, even to an operator with only little training inbacteriology, the participation of several organisms, since thedifferent microcolony sizes, as can be seen from FIG. 4, alreadyindicate three different microorganisms. Under higher magnification, themorphology of the individual bacteria (for example cocci or rods) canalso be taken into account. However, this represents only a possibleadditional aid for the person carrying Out the experiment, since in theprocedure the detection is effected by recording of the correspondinginfra-red spectra. FIG. 6 shows the spectra obtained in the infra-redmode of the FT-IR microscope for the microcolony impressions designated1, 2 and 3 in FIG. 5. Via the characteristic sample curve, all spectraindicate the presence of microorganisms. Comparison of the spectralplots shown in FIG. 6 with those from a reference database of 100spectra of bacteria of 40 different species gave a species-accurateidentification for all microcolony impressions on the basis of thelowest D values, calculated from the first differentiations of thespectra in the wavenumber range of 1500-900 cm⁻¹.

EXAMPLES AND REMARKS WITH REGARD TO FIG. 1

(a) For example urine, liquor, blood, foodstuffs, water, etc., ifnecessary after appropriate preparation and dilution

(b) For example agar plate with added blood, peptone or the like, ifappropriate selective or elective medium

(c) For example 20°-40° Celsius, pH 4-9, for, for example, 4-10 hours orabout 8-20 generation times of the microorganisms

(d) For example stamp impression on ZnSe, BaF₂ discs, polymer filmaluminium platelets, polished steel platelets, etc.

(e) For example manual, visual or automated scanning of the samplecarrier, recording and if appropriate electronic storage of impressionposition coordinates, if appropriate using image processing techniqueswhich provide information with respect to number, position and size ofthe colony impressions

(f) For example series, manual or automatically controlled movement tothe impression coordinates determined in the previous step, selection ofa suitable aperture in the range of about 10-200 μm and recording of thespectra in the wavenumber region of, for example, 5000-500 in the midinfra-red

(g) For example optical evaluation of the spectra by the person carryingout the experiment, calculation of correlation coefficients to referencespectra, multi-variance analysis of the spectra, etc.

(h) If appropriate, identification by comparison with reference data

(i) Taking account of sample dilution etc., if appropriate, additionalresults such as identification, colony shape, etc.

What is claimed is:
 1. A method for rapid detection of microorganisms ina sample, the method comprising the steps of:a) applying the sample to asurface of an essentially solid culture carrier in a dilution whichenables growth of microorganisms in locally separate microcolonies, b)incubating the sample of step a) to form locally separated microcoloniesof 50 to 4,000 microorganism cells, c) transferring a region of thesurface of the culture carrier to a surface of an optical carrier stampwithout changing relative geometrical locations of the transferredmicrocolonies with respect to each other, the stamp surface being atleast one of transparent and reflective in a desired spectral region, d)positioning the optical carrier stamp under an infra-red microscope, e)detecting the transferred microcolonies using an optical mode of theinfra-red microscope, f) determining relative geometrical locations ofthe transferred microcolonies on the stamp, g) sequentially recording IRspectra of the transferred microcolonies, and h) comparing the recordedIR spectra with each other and with a spectra reference file to rapidlydetect microorganisms.
 2. A method for the rapid identification ofmicroorganisms in a sample, comprising the steps of:a) applying thesample to a surface of an essentially solid culture carrier, in adilution which enables growth of microorganisms in locally separatemicrocolonies, b) incubating the sample of step a) for at least 6generation times to form locally separated microcolonies of 50 to 4,000microorganism cells, c) transferring a region of the surface of theculture carrier having microcolonies to a surface of an optical carrierstamp without changing relative geometrical locations of the transferredmicrocolonies with respect to each other, the stamp surface being atleast one of transparent and reflective in a desired spectral region, d)positioning the optical carrier stamp under an infra-red microscope, e)detecting the transferred microcolonies using an optical mode of theinfra-red microscope, f) determining the relative geometrical locationsof the transferred microcolonies on the stamp, g) sequentially recordingIR spectra of the transferred microcolonies using the infra-redmicroscope in at least one of a transmission and a reflection mode, themicroscope having apertures in a diameter range of 10-200 μm, h)comparing the recorded IR spectra with each other and with a spectrareference file to rapidly identify the microorganisms.